The role of chromatin structure in modulating the functions of DNA in transcription, replication, recombination and repair is becoming increasingly apparent. Nucleosome positioning has been suggested to be a possible mechanism in determining the function of cis-acting elements in eukaryotic cells. Previously, we showed that movement of a cis-acting element necessary for yeast replication origin function into the center of a nucleosome markedly diminished its accessibility to trans-acting factors. Similarly, we showed that a yeast repressor organized positioned nucleosome abutting its operator, placing the TATA box in the center of the nucleosome, and suggested that this might be the mechanism of repression of the a-cell specific genes which are repressed by the alpha2 repressor. We have extended these studies, showing that a positioned nucleosome is located 13-16 bp downstream of the alpha2 operator in a plasmid and two genomic contexts. The precision of location of the nucleosomes, together with other considerations, suggested to us that alpha2 might interact with a nucleosome constituent to organize chromatin structure. We have confirmed this supposition, showing that amino-terminal region deletions of histone H4, or certain point mutant substitutions in the same region, lead to abolition of the precise positioning observed for wild type histone H4. As suggested by this result, we show that the expression of a STE6-beta- galactosidase reporter gene was derepressed in a-cells when H4 mutants that abolish positioning were present. Alpha2 forms a heterodimer with al, another homeodomain containing protein, to bind to operators that repress haploid specific gene expression in diploid yeast cells. Methylation interference experiments have now shown that protein-DNA contacts span three successive major and two minor grooves of the nucleic acid. We have investigated the sequence requirements for this interaction and find that the minor groove contacts in the middle of the operator can be eliminated without affecting binding in vitro. We have studied the regulation of the HSP82 gene during sporulation; the HSE element appears to confer diploid specificity for expression while the further upstream sequences are necessary for high level transcription. A second major interest in this project is the relationship of nucleosome positioning and chromatin structure to transcription. We have studied both POL II and POL III genes, including tRNA and 5S rRNA for the latter enzyme. Results suggest that there is a competition in the cell between transcription factors and histones -- for POL III factors and tRNA genes, transcription wins but for POL III and 5S RNA or POL II, positioned nucleosomes can abolish transcription. Collaborative studies of higher order chromatin structure and the crystal structure of a nucleosome core particle containing unique sequence DNA continue.